The use of nanoparticles has increased in consumer products in recent decades; however, concerns regarding their safety remain. Zinc oxide is used in sunblocking and may generate free radicals in response to UV illumination, leading to DNA damage and an immunological response. With high‐resolution, high‐contrast imaging in biological tissue, multiphoton microscopy is able to separate nanoparticles signals from endogenous fluorophores. It has been proven to be very useful in imaging penetration of zinc oxide nanoparticles in skin and in combination with fluorescence lifetime imaging microscopy study cellular function as well. This chapter aims to review the use of these imaging techniques in studying the uptake and distribution of nanoparticles in skin and liver. Due to the questionable clinical use and possible toxicity of nanoparticles, it is important to study their pharmacokinetics. Some nanomaterials have been identified as relatively toxic to humans and a few metal nanoparticles have been reported to penetrate and be detected in blood. Multiphoton microscopy has high resolution and is able to visualize nanoparticles, due to their optical properties, in vivo. The addition of fluorescence lifetime imaging makes it possible to measure the physiochemical environment, with outputs that can be statistically analyzed, posing an advantage over fluorescence intensity imaging only.